Membrane Docking of the Synaptotagmin 7 C2A Domain: Electron Paramagnetic Resonance Measurements Show Contributions from Two Membrane Binding Loops

The synaptotagmin (Syt) family of proteins plays an important role in vesicle docking and fusion during Ca²⁺-induced exocytosis in a wide variety of cell types. Its role as a Ca²⁺ sensor derives primarily from its two C2 domains, C2A and C2B, which insert into anionic lipid membranes upon binding Ca²⁺. Syt isoforms 1 and 7 differ significantly in their Ca²⁺ sensitivity; the C2A domain from Syt7 binds Ca²⁺ and membranes much more tightly than the C2A domain from Syt1, at least in part because of greater contributions from the hydrophobic effect. While the structure and membrane activity of Syt1 have been extensively studied, the structural origins of differences between Syt1 and Syt7 are unknown. This study used site-directed spin labeling and electron paramagnetic resonance spectroscopy to determine depth parameters for the Syt7 C2A domain, for comparison to analogous previous measurements with the Syt1 C2A domain. In a novel approach, the membrane docking geometry of both Syt1 and Syt7 C2A was modeled by mapping depth parameters onto multiple molecular dynamics-simulated structures of the Ca²⁺-bound protein. The models reveal membrane penetration of Ca²⁺ binding loops 1 (CBL1) and 3 (CBL3), and membrane binding is more sensitive to mutations in CBL3. On average, Syt7 C2A inserts more deeply into the membrane than Syt1 C2A, although depths vary among the different structural models. This observation provides a partial structural explanation for the hydrophobically driven membrane docking of Syt7 C2A